Efficient optical plasmonic tweezer-controlled single-molecule SERS characterization of pH-dependent amylin species in aqueous milieus

It is challenging to characterize single or a few biomolecules in physiological milieus without excluding the influences of surrounding environment. Here we utilize optical plasmonic trapping to construct a dynamic nanocavity, which reduces the diffraction-limited detection volume and provides reproducible electromagnetic field enhancements to achieve high-throughput single-molecule surface-enhanced Raman spectroscopy (SERS) characterizations in aqueous environments. Specifically, we study human Islet Amyloid Polypeptide (amylin, hIAPP) under different physiological pH conditions by combining spectroscopic experiments and molecular dynamics (MD) simulations. Based on a statistically significant amount of time-dependent SERS spectra, two types of low-populated transient species of hIAPP containing either turn or β-sheet structure among its predominant helix-coil monomers are characterized during the early-stage incubation at neutral condition, which play a crucial role in driving irreversible amyloid fibril developments even after a subsequent adjustment of pH to continue the prolonged incubation at acidic condition. Our results might provide profound mechanistic insight into the pH-regulated amyloidogenesis and introduce an alternative approach for investigating complex biological processes at the single-molecule level.


Supplementary Note 1. 3D-FDTD Simulation
Three-dimensional finite-difference time-domain (3D-FDTD) simulation was performed using Lumerical FDTD SOLUTIONS to simulate the intensity and distribution of electromagnetic field and caluculate the trapping force and trapping potential.A simplified model was constructed consisting of a pair of dimeric AgNP (70 nm) coated on the junction between two silica beads (1.26 μm).The interparticle gap was set as 10 nm.The dielectric properties of Ag and SiO2 were taken from Johnson & Christy database and Palik database, respectively.The refractive index of background fluid was set as 1.33.A 532 nm Gaussian wave and a 1064 nm Gaussian wave with polarization parallel to the AgNP-coated beads dimer was injected along the z-axis.Perfectly matched layer (PML) boundary condition was used, and the mesh size in the interparticle gap was set as 0.2 nm to increase the accuracy of the simulation.An overall mesh setting with an accuracy of 5 was applied for the rest region.

Trapping force simulation
The force exerted on a AgNP in a harmonic electromagnetic field can be derived using the Maxwell Stress Tensor (MST) [1][2][3] .By integrating the MST over a closed cubic surface surrounding the particle, the time-averaged force can be calculated by: Where  is the time averaged optical force,  is the volume of AgNP,  is the force density,  is the unit volume. is the surface surrounding the particle,  is the unit normal vector to the surface, and  ⃖�⃗ is the Maxwell Stress Tensor which can be expressed as: where  and  represent the outer products of electric field and magnetic field,  ⃡ denotes the unit tensor, and  and  are the dielectric permittivity and magnetic permeability of the surrounding medium, respectively.
Since the diameter of AgNP is much smaller than the wavelength of light, the AgNP itself can be treated as a point dipole with polarizability  = ' + '' = 3   −    +2  .In this case, the trapping potential can be calculated by: Supplementary Fig. 10.Distribution of the characteristic peak positions of single-MB events (red), and single-NBA events (blue) in the BiASERS experiment.Therefore, the optimum scaling factor  1  that correlates the calculated frequencies and the experimental wavenumbers of Tyr at +1 charged state under pH 1.0 condition is 0.9770.
Similarly, by substituting the calculated frequencies and the experimental wavenumbers of Tyr at -2 charged state under pH 13.0 condition in the high wavenumber region (1000-2000 cm -1 ) into the equation, we can get: This quadratic function gives a parabolic graph as shown in Supplementary Fig. 13: Supplementary Fig. 13.Residues as a function of scaling factors correlating the calculated frequencies and the experimental wavenumbers of Tyr at -2 charged state under pH 13.0 condition in the high wavenumber region (1000-2000 cm -1 ).
optimum scaling factor  2  that correlates the calculated frequencies and the experimental wavenumbers of Tyr at -2 charged state under pH 13.0 condition is 0.9770.v(C-C) , β-sheet *Assignments are based on References9-11 .